1. Field Of The Invention
This invention relates to electrical devices used in gel electrophoresis and, more particularly, to a circuit for generating a contour-clamped homogeneous electric field that alternates between two orientations.
2. Description Of The Relevant Art
Gel electrophoresis is a known technique for separating macromolecules on the basis of size, charge, or conformation. In practice, a gel containing the macromolecules to be separated is placed within an electric field, and the macromolecules migrate through the gel in response to the field. In earlier systems, the electric field typically was generated by using a single pair of electrodes. However, unless the electric field direction was varied, large molecules (e.g. DNA) would move at the same rate as smaller molecules (a process called reptation). Furthermore, the size of molecules which may be separated are limited when only two electrodes are used.
One attempt to overcome the foregoing limitations includes providing electrode configurations that generate electric fields in alternating orientations. While this solution may increase the size of macromolecules that may be separated, it creates another problem in that the induced electric field is not uniform throughout the gel as a result of gel and buffer load down, and the macromolecules migrate through the gel with a mobility and trajectory that depend on where in the gel the samples are loaded.
The foregoing problems may be overcome by applying to the gel a homogeneous electric field that alternates between two orientations. A homogeneous electric field theoretically is generated by two parallel, infinitely long electrodes. If one electrode is located along the X axis (Y=O), and the other is separated by a fixed distance (Y=A), the potential field between the electrodes is E(XY)=E.sub.O Y/A, where E.sub.O is the voltage applied across the electrodes.
To simulate a homogeneous electric field with a finite system, a plurality of electrodes are arranged in a cartesian plane along a polygonal contour, such as a square or hexagon. The axes designated Y=O and Y=A are aligned with electrodes which are arranged along parallel sides of the polygon. The electrodes along Y=O and Y=A are clamped to the potentials O and E.sub.O, respectively. The remaining electrodes, located at intermediate positions of the polygon, are clamped to intermediate potentials determined by E.sub.Y =E.sub.O Y/A. Thus, positions along the contour are clamped to potentials equal to those generated by two infinitely long electrodes, and the potential field everywhere inside the contour is also equal to that generated by two infinitely long electrodes. An electric field generated in this manner is termed a "contour-clamped homogeneous electric field" (CHEF).
Alternation in the orientation of the electric field is achieved by electronic switching. For example, a square array may generate a reorientation in the electric field of 90.degree., while a hexagonal array may generate reorientation angles of either 120.degree. or 60.degree., depending on the placement of the gel with respect to the hexagon and the assignment of polarity to the electrodes.
One method used to clamp the intermediate electrodes to the desired voltage is to dispose a series of resistors between the electrodes to, in effect, form a voltage divider having multiple nodes from Y=O to Y=A. Each electrode is connected to a node. However, a disadvantage of using a resistor array to set voltages is that voltages at each of the nodes is dependent on variations in resistor values and the error that is inherent in equally valued resistors. Furthermore, currents which flow from and into each node change the node potential unless the current in the resistor is made much larger than the nodal current. This is wasteful and results in inordinate power dissipation.
Another proposed method for establishing node voltages, not necessarily in the prior art, is to drive each electrode individually with drivers constructed from operational amplifiers. However, some electrophoresis applications require voltages of 300 volts or more, and known operational amplifiers have a maximum operating voltage of under 200 volts. In any event, high power operational amplifiers are very expensive, and they draw excessive current loads on the order of 0.4 amp. Thus, not only are these proposed systems expensive to build, but they are expensive to operate.
Finally, the control elements of analog devices used for direct electrode control cannot be conveniently isolated from the high voltage active devices, and this poses a safety problem, in addition to design restraints. A digital interface is needed, and this escalates the cost of the system. Consequently, an efficient, cost-effective, safe, and accurate CHEF generator having a wide operating range has not yet been devised.